L-rhamnose isomerase produced by Pseudomonas stutzeri LL172 is a known enzyme having the following physicochemical properties revealed in Non-patent document 1.
(i) Action
It is an enzyme catalyzing an isomerization reaction from L-rhamnose to L-rhamnulose and isomerization from L-rhamnulose to L-rhamnose. It is known that it also acts on isomerization between D-allose and D-psicose (Non-Patent Document 1), and it is an enzyme that can produce D-allose from D-psicose. Isomerases are named based on a substrate against which it exhibits the highest activity and a monosaccharide with which the enzymes are induced, therefore, an enzyme named the same L-rhamnose isomerase were isolated from E. coli and Bacillus subtilis, and a gene sequence encoding the same has been reported.
(ii) Substrate Specificity
L-rhamnose and L-rhamnulose are used as a substrate. Other than these, L-lyxose and L-xylulose, L-mannose and L-fructose, D-ribose and D-ribulose, D-allose and D-psicose are used as a substrate.
(iii) Working pH and Optimal pH
The working pH thereof ranges from 7.0 to 10.0 and the optimal pH thereof is 9.0.
(iv) pH Stability
It is stable within a pH range of from 6.0 to 11.0 in the case where it is kept at 4° C. for 1 hour at various pH values.
(v) Working Temperature and Optimal Temperature
The working temperature thereof ranges from 40 to 65° C. and a temperature at which the enzyme exhibits the highest activity in the case where a reaction time is 10 minutes is 60° C.
(vi) Temperature Stability
It is stable at 40° C. for 10 minutes and 90% or more of the activity thereof remains even at 50° C. for 10 minutes.
(vii) Effect of Chelating Agent
The activity thereof is hardly inhibited even if it is allowed to coexist with EDTA or EGTA, which is a chelating agent, during the measurement of the activity.
(viii) Effect of Metal Ion
About 30% of the activity thereof is inhibited by 1 mM cobalt ion.
(ix) Molecular Weight by the SDS-PAGE Method
It is about 43,000.
The present inventors revealed a gene sequence (SEQ ID NO: 3) encoding L-rhamnose isomerase produced by Pseudomonas stutzeri and separately applied for a patent (Patent document 1). As of the date, L-rhamnose isomerase had been isolated from a variety of microorganisms and also the gene sequence encoding the same had been reported, however, there was no report that L-rhamnose isomerase derived from these reacted with D-psicose thereby to produce D-allose.
When the present inventors determined a gene sequence encoding L-rhamnose isomerase produced by a bacterium (Pseudomonas stutzeri LL172) isolated from the soil, it was revealed that the gene sequence does not show homology to any of the gene sequences which have been reported so far and is a novel gene on a gene basis as well as a protein basis (see FIGS. 4 to 6). By utilizing this sequence, an enzyme is produced in a large amount by using genetic engineering and the production of a rare sugar can be carried out using the produced enzyme, or this sequence can be applied to the use using various genetic engineering techniques other than this. Further, the present inventors advanced the studies and revealed that L-rhamnose isomerase produced by Pseudomonas stutzeri has a new catalytic function catalyzing an isomerization reaction of a sugar, which had not been found so far.
The protein described in Patent document 1 is a protein as follows:
(1) a protein comprising an amino acid sequence represented by SEQ ID NO: 4; or
(2) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 and having an L-rhamnose isomerase activity; and
(3) the L-rhamnose isomerase activity is specified by the following physicochemical properties.
(i) Action
It catalyzes an isomerization reaction represented by any of the thick black lines in FIG. 7, FIG. 8 and FIG. 9.
(ii) Working pH and Optimal pH
The working pH thereof ranges from 7.0 to 10.0 and the optimal pH thereof is 9.0.
(iii) pH Stability
It is stable within a pH range of from 6.0 to 11.0 in the case where it is kept at 4° C. for 1 hour at various pH values.
(iv) Working Temperature and Optimal Temperature
The working temperature thereof ranges from 40 to 65° C. and a temperature at which the enzyme exhibits the highest activity in the case where a reaction time is 10 minutes is 60° C.
(v) Temperature Stability
It is stable at 40° C. for 10 minutes and 90% or more of the activity thereof remains even at 50° C. for 10 minutes.
(vi) Effect of Chelating Agent
The activity thereof is hardly inhibited even if it is allowed to coexist with EDTA or EGTA, which is a chelating agent, during the measurement of the activity.
(vii) Effect of Metal Ion
About 30% of the activity thereof is inhibited by 1 mM cobalt ion.
(viii) Molecular Weight by the SDS-PAGE Method
It is about 43,000.
In a conventional production method using an enzymatic reaction method involved in the conversion of D-psicose to D-allose according to the present inventors, in the case where a bioreactor is used, the reaction temperature in the bioreactor is important. That is, when the reaction temperature in the bioreactor is low, contamination with microorganisms during the reaction is caused, and not only is the enzymatic activity decreased, but also the purity or the yield of the product is significantly decreased. Due to this, an industrially important object to be achieved is to carry out a reaction by raising the reaction temperature. Further, in order to maintain the temperature in the bioreactor at room temperature, there is a need to cool the bioreactor in the case of industrial implementation. Energy to be used for cooling the bioreactor is large, which leads to an increase in the cost. From this viewpoint, it becomes an important object for the production of D-allose to raise the reaction temperature.
Patent Document 1: WO 2004/063369
Patent Document 2: JP-A-2002-17392
Non-Patent Document 1: “Journal of Fermentation and Bioengineering”, Vol. 85, pp. 539 to 541 (1998)